6 research outputs found
The initiator methionine tRNA drives secretion of type II collagen from stromal fibroblasts to promote tumor growth and angiogenesis
Summary:
Expression of the initiator methionine tRNA (tRNAi
Met)
is deregulated in cancer. Despite this fact, it is not
currently known how tRNAi
Met expression levels influence
tumor progression. We have found that tRNAi
Met
expression is increased in carcinoma-associated
fibroblasts, implicating deregulated expression of
tRNAi
Met in the tumor stroma as a possible contributor
to tumor progression. To investigate how elevated
stromal tRNAi
Met contributes to tumor progression,
we generated a mouse expressing additional copies
of the tRNAi
Met gene (2+tRNAi
Met mouse). Growth
and vascularization of subcutaneous tumor allografts
was enhanced in 2+tRNAi
Met mice compared with
wild-type littermate controls. Extracellular matrix
(ECM) deposited by fibroblasts from 2+tRNAi
Met
mice supported enhanced endothelial cell and fibroblast
migration. SILAC mass spectrometry indicated
that elevated expression of tRNAi
Met significantly
increased synthesis and secretion of certain types of
collagen, in particular type II collagen. Suppression
of type II collagen opposed the ability of tRNAi
Metoverexpressing
fibroblasts to deposit pro-migratory
ECM. We used the prolyl hydroxylase inhibitor ethyl-
3,4-dihydroxybenzoate (DHB) to determine whether
collagen synthesis contributes to the tRNAi
Met-driven
pro-tumorigenic stroma in vivo. DHB had no effect
on the growth of syngeneic allografts in wild-type
mice but opposed the ability of 2+tRNAi
Met mice to
support increased angiogenesis and tumor growth.
Finally, collagen II expression predicts poor prognosis
in high-grade serous ovarian carcinoma. Taken
together, these data indicate that increased tRNAi
Met
levels contribute to tumor progression by enhancing
the ability of stromal fibroblasts to synthesize and
secrete a type II collagen-rich ECM that supports
endothelial cell migration and angiogenesis
Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion
Developing cell identification methods using atomic force microscopy
This body of work describes the development of a non-invasive and label-free method for characterization of cell surface markers. The motivation for such a method is the ability to measure cells whilst maintaining function, minimizing contamination and disturbance but enabling downstream applications. The technique would impact on life sciences applications including; phenotype identification of both individual and populations of cells, dynamic measurement of cellular response and monitoring cell-microenvironment interactions. The method described centers on molecular recognition interactions which are associated with specific binding forces. These specific forces can be measured in a highly sensitive manner using force instruments. In this study atomic force microscopy (AFM) was employed because of its powerful capability of highly sensitive force measurement at a nanoscale spatial resolution. The objective to develop a force based method for characterization of cell surface molecules may be considered in more specific aims; the development of a functional AFM probe for identification of specific molecules and establishment of quantitative measurement of surface markers. The probe developed has a colloidal geometry which encourages multivalent binding due to greater contact areas, which can reveal presence on cells in just few measurements. On non-deformable surfaces few interactions occur and regular force increments and probability of unbinding indicate presence of target molecules. With multivalent interactions on deformable samples other variables of adhesion indicate identification of interactions; namely distance of total separation, total peaks of unbinding and energy for total separation. With these variables, the identity of HeLa and HFF1 cells was indicated by cluster of differentiation markers 24, 44 and 98 in a semi-quantitative manner. Additionally individual mesenchymal stems cells are identified by the presence of cluster of differentiation marker 90 and dynamic measurement of Human Leukocyte Antigen. Single-cell force spectroscopy was employed to investigate cellular binding to cancerous matrices to gain greater understanding of tumour angiogenesis. Total internal reflection fluorescence microscopy was employed to inform the experimental setting of contact area and sampling density. The method developed illustrates the potential of force based measurement for label-free, non-invasive measurements on cells. Further development and automation may allow the dynamic measurement of multiple markers. This would allow for a number of applications; the identification of true stem cell clones which is of great importance for stem cells therapies, for monitoring of differentiation, where both short and long term activations could be investigated
Bericht der Mobil Oil AG ueber das Geschaeftsjahr vom 1.1.1989 bis 31.12.1989
Available from Mobil Oil A.G., Hamburg (DE) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman
Creating “Living” Polymer Surfaces to Pattern Biomolecules and Cells on Common Plastics
Creating
patterns of biomolecules and cells has been applied widely
in many fields associated with the life sciences, including diagnostics.
In these applications it has become increasingly apparent that the
spatiotemporal arrangement of biological molecules in vitro is important
for the investigation of the cellular functions found in vivo. However,
the cell patterning techniques often used are limited to creating
2D functional surfaces on glass and silicon. In addition, in general,
these procedures are not easy to implement in conventional biological
laboratories. Here, we show the formation of a living poly(ethylene
glycol) (PEG) layer that can be patterned with visible light on plastic
surfaces. This new and simple method can be expanded to pattern <i>multiple</i> types of biomolecule on either a previously formed
PEG layer or a plastic substrate. Using common plastic wares (i.e.,
polyethylene films and polystyrene cell culture Petri-dishes), we
demonstrate that these PEG-modified surfaces have a high resistance
to protein adsorption and cell adhesion, while at the same time, being
capable of undergoing further molecular grafting with bioactive motifs.
With a photomask and a fluid delivery system, we illustrate a flexible
way to immobilize biological functions with a high degree of 2D and
3D spatial control. We anticipate that our method can be easily implemented
in a typical life science laboratory (without the need for specialized
lithography equipment) offering the prospect of imparting desirable
properties to plastic products, for example, the creation of functional
microenvironments in biological studies or reducing biological adhesion
to surfaces